C9 Helices in Crystals of αβ Hybrid Peptides and Switching

Letter pubs.acs.org/OrgLett

C11/C9 Helices in Crystals of αβ Hybrid Peptides and Switching Structures between Helix Types by Variation in the α‑Residue Krishnayan Basuroy, Vasantham Karuppiah, and Padmanabhan Balaram* Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560012, India S Supporting Information *

ABSTRACT: Close-packed helices with mixed hydrogen bond directionality are unprecedented in the structural chemistry of α-polypeptides. While NMR studies in solution state provide strong evidence for the occurrence of mixed helices in (ββ)n and (αβ)n sequences, limited information is currently available in crystals. The peptide structures presented show the occurrence of C11/C9 helices in (αβ)n peptides. Transitions between C11 and C11/C9 helices are observed upon varying the α-amino acid residue.

anonical helical structures, such as the 310 and α-helices found in proteins and polypeptides composed of α amino acids, are characterized by intramolecular hydrogen bonds that run in the same direction.1 The two major helix types have hydrogen bonds of the type COi···HNi+n (n = 3 for the 310 helix and n = 4 for the α-helix), which require orientation of peptide units in the same direction, resulting in the development of a macroscopic dipole moment, with a positive end at the Nterminus and negative end at the C-terminus.2 Close-packed helical structures with mixed hydrogen bond directionality in which alternating CO···HN hydrogen bonds run in opposite directions are unprecedented in the structural chemistry of αpolypeptides. The π(L,D) helix proposed for the gramicidin A sequence, where amino acid chirality alternates, has C16 and C14 hydrogen bonds in opposite directions but has a channel running through the body of the helix.3 The discovery of new hydrogen bonding patterns in polypeptide helices followed rapidly after the observation that helices with both hydrogen bond types COi···HNi+n and NHi···COi+n are possible in poly-β-peptides, which have an additional atom inserted into the backbone.4 The novel C14 helix in which successive hydrogen bonds of type NHi···COi+2 are formed was the first member of this class to be structurally characterized in solution and the solid state.5 Seebach and coworkers provided the first NMR evidence for helices with mixed hydrogen bonding patterns of the type C12/C10/C12 (12/10/12 mixed helix) in their study of oligo-β-peptides with alternating substitution patterns (β2 and β3).5c,6 Theoretical calculations by the groups of Hofmann7 and Wu8 provided a comprehensive evaluation of the energetics of helical structures in sequences containing β residues, permitting an assessment of relative stabilities of helices stabilized by unidirectional and mixed hydrogen bonding patterns. Most importantly, Wu and Wang

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recognized that mixed helices are preferred in sequences with alternating chirality of the β-residues.8b In considering the “chirality” of β-residues derived by backbone homologation of L(S)-α-amino acids, it must be noted that the configuration specified at the asymmetric center is R.4b Molecular dynamics simulations have been used to compute the enthalpy difference between right-handed C12/C10 helix and left-handed C14 helix for an oligo-β-peptide sequence.9 Kessler and co-workers established the C12/C10/C12 helix in an oligo-β-peptide, containing an alternating sequence of the unsubstituted residue β-homoglycine (β-hGly) and a sugar amino acid in which torsional freedom about the Cβ−Cα (θ) bond was constrained by a furanoid sugar ring.10 In an extensive series of investigations Sharma, Kunwar, and co-workers have characterized, by NMR, the C12/C10 mixed helix in oligo-β-peptides containing a sugar amino acid, lacking covalent backbone constraints, in sequences with alternating residue chirality.11 These authors demonstrated the occurrence of C11/C9 mixed helices in hybrid αβ sequences, containing Clinked carbo-β3-amino acids.12 An ab initio MO study provides an indication of the intrinsic potential for formation of C11/C9 mixed helices in hybrid αβ sequences.13 The stereochemical patterning approach has been advanced for the design of novel helices in β-peptides and hybrid αβ sequences.14 While studies in solution provide strong evidence for the occurrence of mixed helices in oligo-β-peptides and hybrid αβ sequences, limited information is currently available in the crystalline state. Reported peptide crystal structures have been largely confined to short sequences of 3−4 residues, permitting characterization of isolated C11/C9 structures in αβ peptides15 and two examples of C12/C10 structure in αγαα and αγαγ Received: July 24, 2014 Published: August 21, 2014 4614

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sequences16. Recently, Lee et al. have described mixed C11/C9 helices in peptides with the alternating D-Ala−cis-ACHC sequences [in the cis-ACHC residue the Cα−Cβ torsion angle θ is constrained by covalent backbone cyclization].17 During the course of studies of hybrid peptides containing the β2,2disubstituted residue β2,2Ac6c (1-aminomethylcyclohexanecarboxylic acid), we observed formation of the mixed C11/C9 structure in the tripeptide Boc-Aib-β2,2Ac6c-Aib-OMe.15d Interestingly, in this example both of the 310/α-helix promoting Aib (α-aminoisobutyric acid) residues adopted the rarely observed semiextended polyproline (PII, ϕ ≈ −60°, ψ ≈ 120°; PII′, ϕ ≈ 60°, ψ ≈ −120°)-like conformations.18 Extension of this sequence to 4 (αβαβ) and 5 (αβαβα) residue sequences yielded incipient C11 helices with conventional hydrogen bond directionality.15d In order to facilitate mixed helix formation by promoting PII conformation at the α residues, we turned to (αβ)n sequences containing alternating chiral α-amino acid and β2,2Ac6c residues. We describe in this report the crystallographic characterization of two tripeptides, Boc-Leu-β2,2Ac6c-Aib-OMe (1) and Boc-Leuβ2,2Ac6c-Leu-OMe (2); one tetrapeptide, Boc-[Leu-β2,2Ac6c]2OMe (3); and one hexapeptide, Boc-[Leu-β2,2Ac6c]3-OMe (4) (Figure 1). The tripeptides Boc-Leu-β2,2Ac6c-Aib-OMe (1) and

Figure 2. Molecular conformations of peptides in crystals. (a) Boc-Leuβ2,2Ac6c-Aib-OMe (1). (b) Boc-Leu-β2,2Ac6c-Leu-OMe (2). (c) Boc[Leu-β2,2Ac6c]2-OMe (3). (d) Boc-[Aib-β2,2Ac6c]2-OMe.15d

Figure 1. Structures of peptides. (a) Boc-Leu-β2,2Ac6c-Aib-OMe 1. (b) Boc-Leu-β2,2Ac6c-Leu-OMe 2. (c) Boc-[Leu-β2,2Ac6c]2-OMe 3 and Boc-[Leu-β2,2Ac6c]3-OMe 4.

Boc-Leu-β2,2Ac6c-Leu-OMe (2) crystallized in the orthorhombic space group P212121 with one peptide molecule in the crystallographic asymmetric unit (Supplemental Table S1). In both cases, mixed C11/C9 conformations are observed (Figure 2a,b). Extension to the tetrapeptide, Boc-[Leu-β2,2Ac6c]2-OMe (3), revealed the persistence of the mixed C11/C9 hydrogenbonded conformation (Figure 2c). This is in sharp contrast to the analogous tetrapeptide, Boc-[Aib-β2,2Ac6c]2-OMe (3), in which Leu (1) and Leu (3) are replaced by Aib residues (Figure 2d) and two consecutive C11 hydrogen bonds are observed.15d The tetrapeptide 3 crystallized in the orthorhombic space group P212121 with one peptide molecule and one co-crystallized solvent molecule (CHCl3) in the crystallographic asymmetric unit (Supplemental Table S2). Encouraged by these observations, we turned to an examination of longer (Leu-β2,2Ac6c)n sequences. Diffraction quality single crystals in the monoclinic space group C2 were obtained for the hexapeptide Boc-[Leu-β2,2Ac6c]3-OMe (4) containing two peptide molecules in the crystallographic asymmetric unit along with one water molecule (Supplemental Table S2). Both molecules fold into a right-handed mixed C11/C9 helix containing four intramolecular hydrogen bonds, C11/C9/ C11/C9 (Figure 3). The relevant backbone torsion angles are listed in Supplemental Table S3. The backbone torsion angles

Figure 3. Molecular conformations of (αβ)n C11/C9 and C11 helices in the peptide sequences (a) Boc-[Leu-β2,2Ac6c]3-OMe (4) and (b) Boc[Aib-β2,2Ac6c]2-Aib-OMe,15d respectively. (c, d) View of the backbone conformations of the two symmetry independent molecules in the crystallographic asymmetric unit of the hexapeptide Boc-[Leuβ2,2Ac6c]3-OMe (4).

reveal that all six Leu residues, in the two independent molecules, adopt semiextended polyproline-like conformations (hydrogen bond parameters for all the peptides are listed in Supplemental Table S4). The structure of the hexapeptide 4 may be compared with a related αβαβα pentapeptide,15d containing Aib residues, which forms a C11 helical structure containing three successive C11 (4 → 1) hydrogen bonds. The crystal structures described in this report provide definitive experimental support for the conformational requirement (PII) at the α-residues in mixed helices. The β-residue necessarily requires a gauche conformation 4615

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(g+/g−) about the Cβ−Cα (θ) bond, depending on the chirality at the α-residue, in order to form hydrogen bonds with alternating directionality. Figure 4a provides a distribution of backbone ϕ−ψ values from both α and β residues observed in the crystal structures of short

bonding patterns (Supplemental Tables S3, S5, and S6 provide the listing of torsion angles, bond lengths, and bond angles used for making the model decapeptide). Figure 5 summarizes the

Figure 5. Average backbone torsion angles of the two types of hydrogenbonded 4 → 1 (αβ) C11 turns and the 1 → 2 (βα) C9 turn. a) C11 turn in unidirectional C11 helices. b) C11 turn in alternate directionality C11/C9 helices. c) C9 turn in alternate directional C11/C9 helices (Average backbone torsion angle values are shown).

backbone torsion angles characterizing two distinct C11 turn types and the C9 hydrogen-bonded turn observed in structurally characterized (αβ)n hybrid peptides (Supplemental Tables S7 and S8 provide a listing of available structures). The two possible helix types, the C11 helix and the mixed C11/C9 helix characterized in (αβ)n sequences containing the β2,2Ac6c residue, differ only slightly in the observed values of θ (Cβ−Cα). It may be noted that the θ values in unconstrained β residues lie close to the ideal gauche value of 60°, while somewhat larger values of 80°− 90° were observed in cyclically constrained residues.19 As seen from Figure 3a,b, a transition between these two helix types may be achieved by major changes in the ψ value at the α residues (PII → αR, Δψ ≈ 180°) and ϕ values at the β residue (−90° → +90°, Δϕ ≈ 180°). Transitions between helices of the same handedness do not involve changes in the sign of θ and can be achieved by 180° flips of alternate peptide units in αβ segments, with relatively minor torsion angle change at βα segments. Further studies in solution are necessary to address the question whether a conformational equilibrium is indeed detectable. In (αβ)n sequences where both residues are chiral, mixed C11/C9 helices may be anticipated for sequences with alternating chirality (heterochiral), as demonstrated in earlier NMR studies.12,20 In the case of homochiral sequences, continuous helices (C11 or C14/C15) may be accessible in the absence of local conformational constraints. Indeed, Gellman and co-workers have provided NMR evidence for rapid interconversion between the 11-helix (C11) and 14/15-helix (C14/C15) for a protected octapeptide composed of alternating sequences of (S,S)-trans-2aminocyclopentanecarboxylic acid (ACPC) and L-α-amino acids.21 It is noteworthy that in the octapeptide Boc-[Aibβ3(R)Val]4-OMe a right-handed C14/C15 helix, which is an analogue of the α-peptide α-helix (C 13 ) observed in crystals.22The backbone conformational parameters differ only slightly from those of the continuous C11 helix. Some general features of value in engineering specific helix types in αβ hybrid peptides emerge from these structural results. In achiral (αβ)n sequences, as exemplified by (Aib-β2,2Ac6c)n peptides, both continuous C11 helices and mixed C11/C9 structures are indeed

Figure 4. (a) Scatter plot in ϕ−ψ space for (αβ)n C11 and C11/C9 helices [θ average of β-residues for C11 and C11/C9 helices are 90.6°(9.0°) and 60.5°(4.1°), respectively]. Supplemental Tables S7 and S8 provide the listing of the structures used. (b) Schematic representation of enantiomeric helix types from both C11 and C11/C9 structures. Transitions between helices of the same handedness do not require significant changes in the values of θ. The points correspond to the average values determined from the analysis of the available crystal structures (see Supplemental Tables S7 and S8). (c) A view of the righthanded (αβ)n C11/C9 decapeptide helix, generated using the average backbone geometries (see Supplemental Tables S3, S5, and S6).

peptides containing a mixed C11/C9 hydrogen bonding pattern. In the case of observations made on peptides lacking chiral αresidues, two molecules of opposite handedness related by reflection or inversion symmetry are observed in crystals, characterized by achiral space groups. In such cases, only one set of torsion angles that corresponds to those observed for L-αamino acid conformations has been chosen, resulting in righthanded helix types. For the β-residues the mean value of the torsion angle about the Cβ−Cα (θ) is ∼60°. The ϕ−ψ values for the β-residues cluster closely around 90°, with opposite signs. An alternative helical structure that may be considered for (αβ)n sequences is the continuous C11 helix, with conventional C Oi···HNi+3 intramolecular hydrogen bonds, which is a backbone expanded analogue of the α-polypeptide 310 helix. Cluster plots for observed ϕ−ψ values in (αβ)n C11 helices are also shown in Figure 4a, to permit direct comparison between two helix types. Figure 4b shows a representation of the right- and left-handed C11/C9 and continuous C11 helices on a 3D ϕ−θ−ψ plot. Figure 4c shows a view of a right-handed, hybrid αβ C11/C9 mixed helix generated for a model decapeptide using the average residue geometries derived from crystal structures of the peptides containing a β2,2Ac6c residue, with C11/C9 mixed hydrogen4616

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(5) (a) Seebach, D.; Overhand, M.; Kuhnle, F. N. M.; Martinoni, B.; Oberer, L.; Hommel, U.; Widmer, H. Helv. Chim. Acta 1996, 79, 913. (b) Appella, D. H.; Christianson, L. A.; Karle, I. L.; Powell, D. R.; Gellman, S. H. J. Am. Chem. Soc. 1996, 118, 13071. (c) Seebach, D.; Abele, S.; Gademann, K.; Guichard, G.; Hintermann, T.; Jaun, B.; Matthews, J. L.; Schreiber, J. V.; Oberer, L.; Hommel, U.; Widmer, H. Helv. Chim. Acta 1998, 81, 932. (d) Appella, D. H.; Barchi, J. J.; Durell, S. R.; Gellman, S. H. J. Am. Chem. Soc. 1999, 121, 2309. (e) Appella, D. H.; Christianson, L. A.; Karle, I. L.; Powell, D. R.; Gellman, S. H. J. Am. Chem. Soc. 1999, 121, 6206. (6) (a) Seebach, D.; Gademann, K.; Schreiber, J. V.; Matthews, J. L.; Hintermann, T.; Jaun, B.; Oberer, L.; Hommel, U.; Widmer, H. Helv. Chim. Acta 1997, 80, 2033. (b) Rueping, M.; Schreiber, J. V.; Lelais, G.; Jaun, B.; Seebach, D. Helv. Chim. Acta 2002, 85, 2577. (7) (a) Möhle, K.; Günther, R.; Thormann, M.; Sewald, N.; Hofmann, H.-J. Biopolymers 1999, 50, 167. (b) Baldauf, C.; R. Günther, R.; Hofmann, H.-J. Angew. Chem., Int. Ed. 2004, 43, 1594. (c) Baldauf, C.; R. Günther, R.; Hofmann, H.-J. Biopolymers 2005, 80, 675. (d) Schramm, P.; Sharma, G. V. M; Hofmann, H.-J. Biopolymers 2010, 94, 279. (8) (a) Wu, Y.-D.; Wang, D.-P. J. Am. Chem. Soc. 1998, 120, 13485. (b) Wu, Y.-D.; Wang, D.-P. J. Am. Chem. Soc. 1999, 121, 9352. (c) Wu, Y.-D.; Han, W.; Wang, D.-P.; Gao, Y.; Zhao, Y.-L. Acc. Chem. Res. 2008, 41, 1418. (9) Lin, Z.; Timmerscheidt, T. A.; van Gunsteren, W. F. J. Chem. Phys. 2012, 137, 064108. (10) Gruner, S. A. W.; Truffault, V.; Voll, G.; Locardi, E.; Stöckle, M.; Kessler, H. Chem.Eur. J. 2002, 8, 4365. (11) (a) Sharma, G. V. M; Reddy, K. R.; Krishna, P. R.; Ravi Sankar, A.; Narsimulu, K.; Kiran Kumar, S.; Jayaprakash, P.; Jagannadh, B.; Kunwar, A. C. J. Am. Chem. Soc. 2003, 125, 13670. (b) Sharma, G. V. M; Reddy, K. R.; Krishna, P. R.; Ravi Sankar, A.; Jayaprakash, P.; Jagannadh, B.; Kunwar, A. C. Angew. Chem., Int. Ed. 2004, 43, 3961. (c) Sharma, G. V. M; Manohar, V.; Dutta, S. K.; Subash, V.; Kunwar, A. C. J. Org. Chem. 2008, 73, 3689. (12) Sharma, G. V. M; Nagendar, P.; Jayaprakash, P.; Krishna, P. R.; Ramakrishna, K. V. S.; Kunwar, A. C. Angew. Chem., Int. Ed. 2005, 44, 5878. (13) Baldauf, C.; Günther, R.; Hofmann, H.-J. Biopolymers 2006, 84, 408. (14) Mándity, I. M.; Wéber, E.; Martinek, T. A.; Olajos, G.; Tóth, G. K.; Vass, E.; Fülöp, F. Angew. Chem., Int. Ed. 2009, 48, 2171. (15) (a) Tanaka, M.; Oba, M.; Ichiki, T.; Suemune, H. Chem. Pharm. Bull. 2001, 49, 1178. (b) Saavedra, C.; Hernández, R.; Boto, A.; Á lvarez, E. J. Org. Chem. 2009, 74, 4655. (c) André, C.; Legrand, B.; Deng, C.; Didierjean, C.; Pickaert, G.; Martinez, J.; Averlant-Petit, M. C.; Amblard, M.; Calmes, M. Org. Lett. 2012, 14, 960. (d) Basuroy, K.; Karuppiah, V.; Shamala, N.; Balaram, P. Helv. Chim. Acta 2012, 95, 2589. (16) (a) Vasudev, P. G.; Chatterjee, S.; Ananda, K.; Shamala, N.; Balaram, P. Angew. Chem., Int. Ed. 2008, 47, 6430. (b) Guo, L.; Zhang, W.; Guzei, I. A.; Spencer, L. C.; Gellman, S. H. Org. Lett. 2012, 14, 2582. (17) Lee, M.; Shim, J.; Kang, P.; Guzei, I. A.; Choi, S. H. Angew. Chem., Int. Ed. 2013, 52, 12564. (18) Aravinda, S.; Shamala, N.; Balaram, P. Chem. Biodiversity 2008, 5, 1238. (19) (a) Choi, S. H.; Guzei, I. A.; Gellman, S. H. J. Am. Chem. Soc. 2007, 129, 13780. (b) Choi, S. H.; Guzei, I. A.; Spencer, L. C.; Gellman, S. H. J. Am. Chem. Soc. 2008, 130, 6544. (20) Srinivasulu, G.; Kiran Kumar, S.; Sharma, G. V. M; Kunwar, A. C. J. Org. Chem. 2006, 71, 8395. (21) Hayen, A.; Schmitt, M. A.; Ngassa, F. N.; Thomasson, K. A.; Gellman, S. H. Angew. Chem., Int. Ed. 2004, 43, 505. (22) Basuroy, K.; Dinesh, B.; Shamala, N.; Balaram, P. Angew. Chem., Int. Ed. 2012, 51, 8736. (23) Rao, P.; Nagaraj, R.; Rao, C. N. R.; Balaram, P. FEBS Lett. 1979, 100, 244.

stereochemically feasible. The observed switch from the C11/C9 mixed helix to the C11 continuous helix on going from the tripeptides to the tetrapeptides may be attributed to the energy penalty that needs to be paid for the Aib residues to adopt the PII conformation,18 which is a requirement for C11/C9 helix formation. In principle, at the tetrapeptide level two intramolecular hydrogen bonds will be formed in both types of helices. The balance between these two helix types is then tilted by the conformational preferences of the Aib residue, which are determined by intraresidue nonbonded interactions. Hybrid (αβ)n sequences containing chiral α-residues and achiral β residues may provide an attractive model system for studying transitions in peptide helices between structures with unidirectional hydrogen bonds and mixed helices. Designed, model (ββ)n peptides containing alternating chiral and achiral residues may be useful in experimentally probing the distribution of C14 helices (1 → 3, reverse hydrogen bond directionality), C12 helices (4 → 1, conventional hydrogen bond directionality), and C12/C10 structures (mixed directionality). Notably the analogous C10/ C8 structures in all α-polypeptides may be difficult to realize because of the requirement of a cis peptide bond to facilitate the C8 hydrogen bond.23 The present study emphasizes the utility of the constrained βresidue β2,2Ac6c in designing specific types of hydrogen-bonded helical structures in (αβ)n hybrid sequences, by varying the nature of the α residue.

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ASSOCIATED CONTENT

S Supporting Information *

Details of synthetic procedure, X-ray diffraction data, structure solution, and refinement procedures and supplementary tables. This material is available free of charge via the Internet at http:// pubs.acs.org. CCDC deposition numbers for the peptides are 988391 (1), 988392 (2), 988393 (3), and 988394 (4), which contain the supplementary crystallographic data for this paper. These data can be obtained free of charge from the Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_ request/cif.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported under the DBT-IISc partnership program. V.K. was supported by a UGC D. S. Kothari postdoctoral fellowship. We thank Dr. S. Raghothama for the 13 C NMR spectra.

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REFERENCES

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